Abstract

Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited antibodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and N-glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc−/− mice exhibit glycemic dysregulations and pancreatic β-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities, and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies.

Introduction

Pancreatic islet allotransplantation is a realistic alternative or complement to insulin therapy in type 1 diabetes (T1D) to prevent serious long-term complications but is limited by the lack of pancreas. Pig pancreas remains a promising complementary islet source. Reproducible evidence exists on the long-term therapeutic benefit of islet xenotransplantation in pig to nonhuman primate preclinical models, using encapsulation for islet immunoprotection (1,2) or using immunosuppressant treatments (3–6). Methods for pig islet purification have significantly improved, in particular for neonatal pig islet-like cell clusters (NPCCs), which are easy to isolate and functional in vivo in the long-term (4,6,7). However, strong humoral response to pig antigens, especially against the α-(1,3)-galactose (GAL) sugar, was detected in nonhuman primates after grafting of neonate (2) and adult (1) pig islets, even encapsulated in alginate hydrogel, and may even affect encapsulated islet survival (1). Humans have lost expression of α-1,3 galactosyl transferase (GGTA1) enzyme generating the GAL epitope (8) and produce natural and elicited antibodies to GAL (9). Among non-GAL, the N-glycolylneuraminic acid (Neu5Gc) sugar is also a major pig xenoantigen (10). Humans have also lost the ability to synthesize Neu5Gc from the N-acetylneuraminic form, after mutation of cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) (11). Pig islets express Neu5Gc, which reacts with human natural antibodies (12,13), and Neu5Gc knockout (KO) mice reject pancreatic islets from wild-type (WT) counterparts (14).

Using transcription activator-like effector nucleases (TALEN), we obtained a double-knockout (DKO) pig line for the CMAH and GGTA1 enzymes. However, CMAH−/− mice exhibited higher fasting blood glucose and insulin than WTs, associated with mild glucose intolerance and significant β-cell dysfunctions, including higher insulin secretion by isolated islets in response to glucose (15). As a prerequisite for xenocell therapy for T1D and to study the metabolic effects of CMAH KO in a species more closely related to humans than mice, we analyzed glucose homeostasis in our DKO adult pigs and insulin secretion of isolated DKO NPCCs.

Research Design and Methods

Animals and Ethical Statements

DKO pigs were obtained by targeting the swine CMAH gene using TALEN on the GGTA1−/− background (obtained from Dr. D.H. Sachs, Massachusetts General Hospital, Boston, MA) and by somatic cell nuclear transfer (SCNT) (Supplementary Data). WT pigs were Landrace × Large-White (EARL Pont Romain, Surzur, and ANSES, Ploufragan, France). Adult and neonate pigs were males, respectively, 5–6 months old weighing 37.90 ± 3.47 kg and 3–10 days old weighing 1–3 kg. CMAH−/− (B6.129 × 1-Cmahtm1Avrk/J; The Jackson Laboratory, Bar Harbor, ME) and control mice (C57BL/6; Janvier Laboratories, Le Genest-Saint-Isle, France) were 8–12 weeks old. Experiments were approved by the Ethics Committees and performed in accordance with relevant Italian (DGL116/92) and French regulations (2001-464 and 2013-118, Approval 01074.01/02). All efforts were made to minimize animal suffering and to restrict the number of experimental animals.

Analysis of adult DKO pig pancreas. A: Histological analyses of formalin-fixed paraffin-embedded pancreatic tissue 4-μm cross-sections stained with hematoxylin-eosin-saffron from one WT pig (right) and one DKO pig (left). B: Immunostaining of insulin (green, Alexa-Fluor 488) and glucagon (red, Alexa-Fluor 555) on 2.5-μm pancreatic tissue sections from one WT pig (right) and one DKO pig (left). Images (Zen 1.0 software; Carl Zeiss MicroImaging, Inc., Jena, Germany) are representative of the pancreas of 4 DKO and 4 WT pigs, and microscopic scales are indicated on the pictures. C–E: For islet number and size, at least 100 islets were investigated per animal (4 DKO and 4 WT pigs). For each animal, 5.28 mm2 of the head, the body, and the tail of the pancreas tissue were observed. C: Mean islet size (×103 µm2) in DKO and WT pigs. D: Percentage of islet area per pancreas area. E: Number of islets per mm2 of pancreatic area. F–H: For insulin and glucagon immunostaining analyses, at least 70 islets of the head, the body, and the tail of the pancreas tissue were analyzed per animal (4 DKO and 4 WT pigs). F: Mean insulin-positive area per islet (×103 µm2). G: Mean glucagon-positive area per islet (×103 µm2). H: Percentage of glucagon area per insulin area. Quantity of insulin (ng) (I) and glucagon (ng) (J) per protein quantity (μg) by Bradford protein assay (Fisher Scientific) of the pancreas tissue (4 DKO and 4 WT pigs). C–H: Image analyses were investigated using the ImageJ software (https://imagej.nih.gov/ij/index.html). All values are expressed as mean ± SEM. Differences between DKOs and WTs are not significant by Mann-Whitney test.

In IVGTT, blood glucose curves were very similar between DKO and WT pigs. Blood glucose peaked 3 min after glucose administration and then dropped to reach the fasting value after 60–90 min (Fig. 3A). The rate of glucose disappearance did not differ (2.42 ± 0.63 mmol/L/min for DKOs and 2.47 ± 0.78 for WTs) (Supplementary Table 1). AUCglu were also almost identical (421.8 ± 46.58 mmol*120 min/L and 435.5 ± 19 for DKOs and WTs, respectively). Serum insulin increased 8–17-fold in the first minutes after glucose injection (Fig. 3B). C-peptide also showed rapid elevation (Fig. 3C). The DKO pig number 3 (DKO3) had a delayed insulin (15 min after glucose administration) and C-peptide (10 min) peak. Consistently, DKO3 presented a moderately elevated AUCglu associated with a slightly lower AIRglu and AUCins than WT and other DKO pigs, but not objectified by a different AUCC-pep (Supplementary Table 1). Glucagon suppression was more rapid and intense in WTs and DKO3 than in the other DKOs (Fig. 3D).

During ITT, blood glucose in DKO pigs dropped rapidly and was similar to controls (Fig. 3E). Glucose disappearance did not differ between pig groups (KITT, 11.36 ± 1.50% for DKOs vs. 10.73 ± 0.82 for WTs) (Supplementary Table 1). C-peptide curves were also similar between pigs, except for the DKO pig number 2 (DKO2) exhibiting a chaotic waning of C-peptide after insulin injection (Fig. 3F).

Pancreatic islets from neonate DKO pigs. A: Extracellular insulin was quantified by ELISA in the supernatant of 50 IEQ NPCCs on day 1 after isolation, after 120 min culture in basal medium (2.8 mmol/L glucose), and after glucose (20 mmol/L) ± theophylline (20 mmol/L) stimulation. The NPCCs from five DKO and six WT pigs were investigated in three replicates for each islet preparation in three independent experiments. The results are given as mean insulin stimulatory index (S.I.) ± SEM for the DKO and WT groups. *P = 0.026; **P = 0.0043. Other differences between the groups were not significant (Mann-Whitney test). B: Five hundred IEQ Si-HPMC–encapsulated NPCCs of DKO, GAL KO, and WT control neonate pigs were grafted subcutaneously in WT or CMAH−/− mice. The serum of grafted mice was tested 15 to 20 days later for the presence of anti-Neu5Gc IgG. The mouse serum was incubated with Neu5Gc+/+ thymocytes from C57BL/6 mouse and phycoerythrin-antibody anti-mouse IgG (BioLegend, San Diego, CA) as per Tahara et al. (14). Analyses were performed by Aria flow cytometry (BD Biosciences). Data were analyzed by FlowJo software. The black histograms represent phycoerythrin-isotype control staining and the gray histograms phycoerythrin-antibody anti-mouse IgG (detecting IgG specific for Neu5Gc). Control pig islets in CMAH−/− mouse (B1), GAL KO pig islets in CMAH−/− mouse (B2), control pig islets in WT mouse (B3), and DKO pig islets in CMAH−/− mouse (B4). One representative experiment of two independent experiments is shown, for a total of two to three in each group.

Discussion

Anti-GAL antibodies have been considered for years as the first obstacle to successful xenotransplantation. Natural anti-Neu5Gc antibodies are likely to be important non-GAL antibodies because they are present in most normal human sera (12,17) and are elicited by challenges with xenogenic tissues (18). Preexisting antibodies present in human serum bind to GAL−/− pig islets (19). Moreover, WT syngeneic pancreatic islets are rapidly rejected by CMAH−/− mice and not by GAL−/− mice (14).

Unlike the CMAH−/− mouse (15), fasting blood glucose and insulin and glucagon secretions appeared normal in adult DKO pigs. Furthermore, DKO pigs seemed to exhibit neither glucose intolerance nor insulin resistance as indicated by changes in blood glucose levels after IVGTT and ITT, respectively (confirmed by HOMA-IR). Although the number of adult control pigs tested in our study was limited, the results with control WT pigs were similar to those with DKOs, and values remained within the physiological range for fasting and IVGTT in pigs (24). After glucose administration, one DKO pig had delayed insulin secretion without this clearly affecting glycemia regulation, probably as glucagon rapidly decreased in this pig.

DKO pigs also displayed preserved islet architecture (islet number, area, volume density, and insulin and glucagon immunostaining). Further studies with a high-energy diet would be helpful to investigate the effect of the CMAH KO on β-cell and islet compensation in response to severe obesity-induced insulin resistance (15).

Furthermore, isolation of NPCCs yielded an IEQs-to-g pancreas ratio similar to that resulting in control pigs. DKO NPCCs were functional ex vivo and exhibited insulin secretion after stimulation with glucose and theophylline. Pig islet Neu5Gc and sialic antigens clearly contribute to pig islet antigenicity (12,13). Also, our results clearly confirmed that anti-Neu5Gc antibodies are induced in CMAH−/− mice by WT encapsulated NPCCs, probably mimicking what is expected in the human context. CMAH editing in our pigs prevented this humoral-specific response, consistent with earlier observations of reduced binding of human antibodies to peripheral blood mononuclear cells from GGTA1/CMAH KO pigs (25). CMAH editing will most likely be necessary when hydrogel-encapsulated pig islets are used for T1D xenotherapy because encapsulation in hydrogel does not fully prevent antisugar humoral responses (1,2).

In conclusion, our study opens the way for the use of DKO pig pancreatic islets in (pre)clinical studies on T1D cell therapy.

Article Information

Acknowledgments. The authors are very grateful to David H. Sachs (Massachusetts General Hospital, Boston, MA) for providing the GAL KO pig. The authors thank Odile Duvaux (Xenothera, Nantes, France) for the scientific discussions.

Funding. This work was supported by Pays de la Loire Region (France) (Xenothera academic program and to A.S.), the Société d’Accélération du Transfert de Technologies Ouest Valorisation (to A.S.), the European Center for Transplantation and Immunotherapy Sciences (ECTIS IHU, Nantes, France), the National Research Agency “Investment Into The Future” programs (ANR-10-IBHU-005, to X.L., and ANR-II-INSB-0014), the European Commission’s Xenome Sixth Framework Programme (LSHB-CT-2006-037377), and the European Seventh Framework Programme “Translink” research program (grant agreement 603049 and to L.L.B.).

Duality of Interest. A.S. is currently an employee of the start-up Xenothera. J.-P.S. and J.-M.B. are cofounders of the start-up Xenothera. No other potential conflicts of interest relevant to this article were reported.